In microscopic digital imaging of specimen, a typical area of interest for a digital scan of a specimen is 6×6 mm. The resolution needed for clinical purposes is typically 5 pixels/μm leading to a total image map of 30000×30000 pixels=900·106 pixels. Using 24-bit colour, this corresponds to an image map of approximately 3 GB. This data can be collected using a motorized microscope with an attached digital imaging device.
When trying to increase speed of microscopic digital imaging, a problem to solve is the transfer rate of image data—the larger image data, the longer time it takes to transfer the image data. Another problem to overcome is the capacity demanding focusing of the focusing system. Acquiring sharp images at these magnifications constitutes a challenge due to the narrow depth of field. The focus surface, the collection of points where an image of the sample is acquired in focus (i.e. the image is sharp), has to be determined with great accuracy.
One way of determining a focus for a point in the sample is by acquiring a set of images of the point in the sample and varying the distance between the sample and the optical system for every image. The image with best focus is selected using for example a relative focus metric.
Let the specimen to be examined extend in an x- and y-direction and have a thickness in a z-direction. In an ideal world with perfect hardware (zero tolerances, straight bearings, no play, etc) and perfect plane smears of the specimen to be examined—the focus surface would be a plane. For a dense smear in a machine with ideal hardware, the fastest way to image an area would be to first determine focus at three different (x, y)-positions spread far apart. Then, using the three resulting (x, y, z)-points, the focus surface would be estimated and all images of this surface could be acquired without further focus determination. However, the focus surface in real digital microscopes using high magnification (e.g. 50× and 100×) is often quite rugged, especially if inexpensive parts are used in the mechanical system. Moreover, this surface structure can change with every slide. The trade-off between focus quality and speed is challenging since on one hand a careful focus determination at each position will give good focus quality but take long time. But, on the other hand, acquiring an image directly at a guessed, predicted, or estimated z-position at each position will not take long but give poor focus quality.
One of the keys to fast image acquisition is to have a good prediction function of the focus surface; the better the prediction, the less time is spent on collecting a sharp image at each position.
A common method to acquire a good z-prediction is to build a focus map in a separate step prior to scanning. The focus map is based on a number of focus positions, which can be determined either manually or automatically. However, these methods are time consuming for complex focus surfaces since the focus map needs to be based on many focus positions in order to give a good prediction.
Thus, there is a need for a prediction method that combines cost-efficiency with high transfer speed and precision.